In 2019, this project provided state-of-the-art research technologies for NIAID's intramural infectious diseases, allergy, and immunology research programs. Technologies developed within the RTB, and outside the NIH, are tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the NIAID intramural program. The technologies supported in the RTB include flow cytometry, confocal microscopy, protein separation, mass spectrometry, peptide synthesis, protein sequencing, and macromolecular X-ray crystallography. Many of these technologies are used in high containment laboratories critical to the Institute's infectious diseases and biodefense research agenda. In addition to technology development, the RTB provides advanced training in all aspects of the technologies in the Branch's portfolio. Flow Cytometry The Flow Cytometry Section (FCS) provides the DIR with application-specific flow cytometric technologies, including instrumentation for high speed cell sorting and multi-color analysis. In addition, the FCS provides training, consultation, method development, and analysis for experiments involving flow cytometry. This mission is accomplished through the efforts of staff with extensive flow cytometry experience and with the use of six cell sorters and eight analyzers. Major goals are to provide access to state-of-the art technologies, to help design and run experiments, to facilitate data interpretation and to provide results that are of consistent high quality. FCS consists of a wide variety of instrumentation and provides a cost- and time-efficient method of obtaining fluorescent-labeled custom antibodies. Resources available include hybridoma expansion; antibody purification from ascites, antisera, and hybridoma supernatant; and coupling of purified antibodies and proteins to various fluorochromes. Biological Imaging The Biological Imaging Section (BIS) makes advanced light microscopy imaging technologies and expertise available to DIR scientists. The section collaborates in experimental design, instrument operation and in increasingly-sophisticated image processing routines to best utilize the power of these cutting-edge technologies. Experienced scientists can use the microscopes unattended with staff standing by for troubleshooting, thus allowing the DIR maximal use of facility infrastructure. BIS offers instrumentation and expertise in advanced light microscopy imaging technologies including confocal, fluorescence, video microscopy, and post-collection quantification and deconvolution. BIS is a resource for Stimulated Emission Depletion (STED) microscopy, multiphoton confocal microscopy (intravital imaging), sample preparation, long time-lapse imaging, the NIAID Imaris licenses, and poster printing. Investigators participate in the collection of their data, either as direct microscope operators or by selecting cells of interest, so that the staff can then collect the data. BIS advises on the most suitable instrument, appropriate labels, sample preparation, and best sampling regimens. BIS advises on the correct use of the most appropriate software analysis modules, as well as the best format for displaying the data. Frequently, the Section works with investigators to prepare images for publication including still images, video sequences, 3D reconstructions and time-lapse animations. Protein Chemistry The Protein Chemistry Section develops project specific applications in the fields of peptide synthesis, N-terminal (Edman) sequencing, protein separation, assay development, and mass spectrometry. The RTB designs qualitative and quantitative physical-chemical and biochemical methods of detection and analysis that are customized to meet the specific research needs of DIR investigators. The Branch is also active in formulating and executing purification strategies for various types of molecules. This can also include customized small-scale sample preparation and enrichment strategies for analysis by mass spectrometry. Another major activity of the RTB involves the development of more efficient and effective methods of sample preparation with a strong orientation towards mass spectrometry to facilitate protein identification work. The Branch collaborates with DIR investigators to develop and validate protocols for protein separation and analysis for specific research programs of interest to the intramural research program. The Mass Spectrometry Facility provides support in the planning and execution of mass spectrometry (MS) projects and cooperates with other labs within the RTB to support analysis and characterization of peptides, proteins, and small bio-molecules. The facility offers all facets of mass spectrometry projects including experimental design, separation of biomolecules, sample preparation, data collection and analysis/interpretation of data. The MS facility focuses on analyzing proteins and peptides, and characterizes small molecules upon request. Genomic Technologies The Genomic Technologies Section (GTS) provides state-of-the-art next-generation sequencing, and other massively parallel genomic technologies to intramural investigators. In addition to managing high-end instrumentation, computers, and protocols involved in genome-wide assays, the GTS offers statistical analysis and bioinformatics for data interpretation. This support involves close collaboration with the investigators in both experimental design and data analysis to best utilize the power of these applications, as well as to convey a better understanding of the technology. GTS develops project-specific research applications for many analysis platforms at the DNA, RNA and protein level. Statistical analysis, data management, and data mining solutions are available as well, including from experimental concept to publication and public data submission. The Section maintains instrumentation and resources for sequencing applications using the Illumina high throughput sequencing (NGS), including RNA-seq, DNA-seq, ChIP-seq, ATAC-seq, Rep-seq, and single-cell RNA profiling and many others. Some microarray applications such as single-cell Western and protein arrays are supported as well. Structural Biology The Structural Biology Section (SBS) provides specialized techniques and scientific expertise that enable DIR scientists to obtain biophysical and structural data for macromolecules. While closely collaborating with DIR researchers, the SBS provides consulting/training, produces pure proteins, performs biophysical analyses, and determines structures of proteins and other macromolecules that are central to the infectious disease and immunology research programs of the DIR. Biophysical analysis and structural biology require expertise in producing large amounts of correctly folded proteins at high-purity, in preparing diffraction-quality crystals, and in determining crystal structures by X-ray methods. With an emphasis on providing training in biochemical, biophysical, and structural methods, the SBS makes it possible for investigators to use structurally based ideas and techniques in their research program.